ABSTRACT SUMMARY Acute lung injury is reported to be one of the most common forms of organ dysfunction in critically injured individuals. Findings from a number of studies suggest that priming occurs at several cellular levels, in the lung and circulating neutrophils, and appears to play significant roles in mediating the increased inflammation associated with this injury. Utilizing a variety of experimental approaches (neutrophil/ macrophage depletion, adoptive transfer of neutrophils, gene specific knock-out/over-expression, in vivo antibody neutralization/ peptide antagonism/ siRNA) in a dual insult (non-pulmonary) model of hemorrhagic shock (Hem) followed by polymicrobial septic challenge (CLP), we have found evidence of `priming both in circulating neutrophils and pulmonary epithelial cells for enhanced pro-inflammatory responsiveness. However, in neutrophils, this has been associated with suppressed apoptosis, while in epithelial cells;a potentiation of apoptosis has been observed. Further, it would appear that epithelial cell `priming'after Hem for localized cytokine/ chemokine expression in the lung is antecedent/ required for macrophage activation and the eventual recruitment of `primed'neutrophils to the lungs of animals subsequently challenge by CLP. The contributions of the endothelia and other localized tissue immune cells, e.g., dendritic cells and/or lymphocytes in this activation process have not been defined. Finally, studies utilizing organ specific delivery of siRNA against extrinsic death receptor protein, Fas, indicate not only that anti-apoptotic approaches appear efficacious but that these approaches also serve to suppress aspects of the local pro-inflammatory response. However, the nature of this protection at cellular levels as well a molecular level is not clear. With this in, mind we propose the following hypothesis, which the mechanisms by which localized delivery of Fas siRNA mediates its'effects are through both inhibition of apoptosis and pro-inflammatory activation of epithelial cells `primed'by shock/Hem. To test this hypothesis, we have designed the following objectives (Aims): AIM 1: We will determine the nature of Fas/FasL mediated signaling/activation of pulmonary epithelial cells, endothelial cells, neutrophils and/or alveolar macrophages to a `priming'insult such as Hem. AIM 2: We will determine the cellular source of FasL epithelial cell injury/ damage. AIM3: We will establish the nature of the inducer of Fas/FasL expression in the lung following shock. AIM 4: We will establish if endothelial cell activation and/or injury are pre- or post- to the activation/'priming'/injury of epithelial cells following shock.PROJECT NARRATIVE Acute lung injury is reported to be the most common form of organ dysfunction encountered in critically ill patients in trauma/ surgical/ medical intensive care units. This study is designed to determine what the contribution of immune (i.e., neutrophil, macrophage, dendritic cell, etc.) and non-immune cell's (i.e., epithelial and endothelial cell) natural cell suicide (apoptotic) program and/or inflammatory response are to the development of lung injury resulting from the combined insult of shock (due to blood loss) followed by septic challenge in the mouse. The contribution of these cells's apoptotic/ inflammatory processes will be examined in this setting using genetically altered mice as well as drug interventions directed at genes or mediators thought to be involved in the regulation of these processes. It is our firm belief that the results of these studies will provide information that not only should allow us to better understand the pathobiology of acute lung injury but also its attenuation.